Preparation of Rabbit Antihuman Albumin Serum and Purification of the IgG Antibodies.

We applied a very simple method,TCA-al-cobol method,to purify human serum albumin.The product is pure albumin by SDS-PAGE.Emulsified with Freund’s complete adjuvant(FCA),it was used to immunize rabbits by a pro-cedure which lasts only five weeks.Specific anti-serum with high titration(1:128)was obtained.

Isolation and Characterization of Rat Liver Polyribosomes.

As the first step of selecting a specific mRNA,we would have polyribosomcs available.Weused ultracentrifugation method to isolate poly-ribosomes from rat liver.The UV spectrum andA260/A280 ratio of this preparation showed thecharacteristics of polyribosomes,so did the sedi-

共培养法分离培养不同遗传背景的小鼠精原干细胞

目的 建立一种简便、高效的小鼠精原干细胞(mouse spermatogonial stem cell, mSSC)分离、培养方法。方法 选取昆明小鼠和C57BL/6小鼠的雄性幼鼠睾丸,通过机械分离和酶消化相结合的方法获得细胞悬液,随后将含有mSSC和支持细胞以及其它细胞的混合悬液以合适的密度接种至培养皿中共同培养,待mSSC形成克隆,但支持细胞还未进入大量增值期时,将mSSC移至饲养层细胞中扩大培养,最后通过特异性分子标志物检测和诱导分化等实验对分离的mSSC进行鉴定。结果 通过将mSSC和支持细胞共同培养的方法建立了mSSC体外分离培养的共培养法,该方法从昆明小鼠和C57BL/6小鼠两种不同遗传背景的幼鼠睾丸中成功分离mSSC。RT-PCR实验结果表明,本研究分离培养的mSSC表达mSSC的特异性分子标志物Plzf、Gfrα-1和Ngn3,使用维甲酸(retinoic acid, RA)诱导后可观察到精子样细胞的出现,使用流式细胞术进行倍体分析表明,诱导后的细胞中有9.52%为单倍体,而未诱导的细胞中检测不到单倍体细胞存在。RT-PCR实验结果表明,诱导后mSSC开始表达精原干细胞分化相关基因Acrosin、Th2b和Sycp3。结论 本研究建立了一种适合不同遗传背景mSSC的体外分离、培养方法,该方法既简化了分离步骤,又节约了培养成本,为广泛开展mSSC的研究提供了条件。

圆头精子症患者的致病基因变异分析4例

目的对4例圆头精子症患者的致病基因进行分析,明确其遗传学病因。方法选取2017年6月至2020年9月在中国人民解放军联勤保障部队第九〇〇医院被确诊为圆头精子症的4例患者作为研究对象,采集其精液和血液样本,进行精子浓度、活力、存活率、形态等常规检测以及精子顶体抗原CD46免疫荧光检测,提取外周血样基因组DNA进行全外显子组测序(WES),对疑似致病变异通过Sanger测序进行验证。结果WES检测显示4例患者均携带DPY19L2基因变异,其中患者1~3携带DPY19L2基因纯合缺失,Sanger测序显示患者3在重组断裂点区域BP2存在断裂,通过非等位基因同源重组造成DPY19L2基因完全缺失。患者1存在第2~22外显子纯合缺失,患者2存在第14~15外显子纯合缺失,患者1和2的缺失既往未见文献报道,患者4为DPY19L2基因杂合缺失,且3′UTR区存在罕见的纯合缺失。结论DPY19L2基因变异可能导致的圆头精子症的发生,其方式符合常染色体隐性遗传,同时也符合基因组病的特点。

Construction of sperm-specific lactate dehydrogenase DNA vaccine and experimental study of its immuno-contraceptive effect on mice

Lactate dehydrogenase C4 (LDHC4) is a key enzyme for sperm metabolism. It is distributed specifically in testis and is highly immunogenic. In this study, two DNA vaccines pVAX1-hLDHC and pVAX1-mLDHC were constructed by inserting coding sequences of human and mice LDHC4 into the eukaryotic ex-pression vector pVAX1. The production of LDHC4 specific antibodies was induced in the sera of vac-cinated mice and the reproductive tract secretions of vaccinated female mice through immunization by mucosal surface instillation. Furthermore, the antibody titer increased with the times of immunization. In the mating experiment, the number of newborns of the vaccinated mice reduced significantly and some immunized female mice even lost the ability to bear any offsprings, suggesting that the difference between the immunized and control mice was statistically significant. Sperm agglutination analysis indicated that both the antisera from immunized mice and the reproductive tract secretions of vacci-nated female mice could agglutinate normal sperms. Results of immunohistochemistry showed that the antibodies present in the sera of immunized mice and the reproductive tract secretions of vaccinated female mice could specifically react with LDHC4 antigen, which mainly locates in the cytoplasm, acrosome membrane externa and acrosome capsule of the sperm. Taken together, our results indicated that the constructed contraceptive DNA vaccines did yield immunocontraceptive effects on mice and this would enable clinical trials in near future.

miRNA-29c在1型糖尿病早期肾病患者中的表达及其诊断价值

目的研究miRNA-29c在1型糖尿病(type 1 diabetes mellitus,T1DM)早期肾病患者中的表达情况,及对早期肾病的诊断价值。方法回顾性选取2019年1月至2022年3月期间于杭州市临安区第一人民医院接受治疗的168例T1DM患者作为研究对象,根据肾病发生情况分为单纯性糖尿病组(122例)及糖尿病肾病组(46例)。采用RT-PCR法检测血清miRNA-29c水平,比较两组的性别、年龄、病程、血清miRNA-29c水平等一般资料,采用Logistic回归分析T1DM患者发生早期肾病的影响因素。采用ROC曲线分析miRNA-29c对于T1DM发生早期肾病的诊断价值。结果早期肾病组的病程、血压(收缩压、舒张压)、糖化血红蛋白(HbA1c)、总胆固醇(TC)、血尿酸高于单纯性糖尿病组,而白蛋白、总胆红素、miRNA-29c(0.82±0.22 vs 1.24±0.33)低于单纯性糖尿病组,差异有统计学意义(P<0.05)。多因素Logistic分析显示:长病程(OR=2.061,95%CI=1.090~3.896)、收缩压(OR=1.143,95%CI=1.023~1.279)、舒张压(OR=1.151,95%CI=1.022~1.298)、高HbA1c(OR=1.317,95%CI=1.049~1.653)、高血尿酸(OR=1.306,95%CI=1.028~1.659)、低miRNA-29c(OR=0.845,95%CI=0.730~0.979)是影响T1DM患者发生早期肾病的危险因素(P<0.05)。ROC曲线分析显示miRNA-29c诊断早期肾病的截断值为0.952,曲线下面积(AUC)为0.863(95%CI:0.801~0.925),敏感度、特异度分别为84.78%、80.33%。结论T1DM早期肾病患者血清miRNA-29c处于低表达状态,是T1DM患者发生早期肾病的影响因素,对于早期肾病具有良好的诊断价值。

一个COL11A1基因新剪接变异导致的先天性高度近视家系的遗传学分析

目的 对1个先天性高度近视家系进行基因变异分析,探讨其致病原因.方法 对先证者进行全外显子组测序,应用Sanger测序验证可疑致病变异,并进行家系共分离分析.结果 基因测序显示先证者COL11A1基因存在c.2556+1G>A剪接位点杂合变异,家系中另3例患者(先证者母亲和两个女儿)均存在c.2556+1G>A杂合变异,表型正常家系成员(先证者父亲、妹妹)均未发现该变异,表型与基因型在家系内共分离.经查阅OMIM、HGMD及Clinvar数据库,c.2556+1G>A变异为未报道过的新变异.根据ACMG遗传变异分类标准与指南,c.2556+1G>A变异符合PVS1+PM2,可以判定为可能致病性变异.结论 COL11A1基因c.2556+1G>A剪接变异可能是该家系患者的致病原因,致病性变异的检出为家系的遗传咨询和产前诊断提供了依据.